Polycystic ovary syndrome (PCOS) is the most common reproductive disorder in women. MicroRNA (miRNA) plays important regulatory roles in PCOS development, but its biological mechanism remains poorly understood. Innovating the visualization methods of miRNA has great significance for studying the genesis and development of PCOS. Here, we report near-infrared (NIR) light-triggerable DNA nanoprobes (termed as LT-DP) for visualizing miRNA-103-associated molecular mechanisms in clinical ovarian granulosa cells (GCs) of PCOS patients. An amplified fluorescence signal of the LT-DP nanoprobes occurs only when the catalytic hairpin assembly amplification reaction is triggered by NIR light and target miRNA, resulting in high sensitivity and specificity of miRNA-103 imaging in human granulosa-like tumor cells (KGN). Attributable to its imaging capability, LT-DP nanoprobes discriminate GCs of insulin resistant (IR) and non-IR PCOS patients with differential miRNA-103 expression. We further investigated the miRNA-103-associated molecular mechanisms and observed that the upregulation of miRNA-103 in PCOS patients promotes the development of ovarian IR by inhibiting the IRS1/PI3K/AKT/GLUT4 signaling pathway. Additionally, the tracking of miRNA-103 expression reveals that metformin (Met) could relieve the ovarian IR by modulating the IRS1/PI3K/AKT/GLUT4 signaling pathway. The LT-DP nanoprobes demonstrate clinical potential and provide a strategy to reveal the disease mechanisms.